Amphoteric liposomes comprising imino lipids

ABSTRACT

The invention concerns lipid assemblies, liposomes having an outer surface comprising a mixture of anionic and cationic moieties; wherein at least a portion of the cationic moieties are imino moieties that are essentially charged under physiological conditions, and their use for serum resistant transfection of cells.

FIELD OF THE INVENTION

The present invention relates to lipid assemblies or liposomes that arecapable of overcoming a lipoprotein mediated uptake blockade. Morespecifically, this invention relates to improvements in liposomescomprising both negatively charged lipids having a carboxylic orphosphate head group and positively charged lipids having imino orguanido moieties or derivatives thereof in the respective polar regions.

BACKGROUND TO THE INVENTION

Liposomes have widespread use as carriers for active ingredients.Neutral or negatively charged liposomes are often used for the deliveryof small molecule drugs, whereas positively charged (cationic) or therecently introduced class of amphoteric liposomes are mainly used forthe delivery of nucleic acids such as plasmids or oligonucleotides.Important examples for cationic liposomes used for the delivery ofnucleic acid cargoes include, but are not limited to Semple et al., Nat.Biotech. (2010) 28:172-176; Akinc et al., Nat. Biotech. (2008)26:561-569; Chien et al., Cancer Gene Ther. (2005) 12:321-328; deFougerolies, Nat. Rev. Drug Discov. (2007) 6:443-453; Kim et al., Mol.Ther. (2006) 14:343-350; Morrissey, Nat. Biotech. (2005) 23: 1002-1007;Peer, Science (2008) 319: 627-630 and Santel, Gene There. (2006) 13:1222-1234. Application of amphoteric liposomes for the delivery ofnucleic acids has been demonstrated in Andreakos et al. Arthritis Rheum.(2009) 60:994-1005.

Amphoteric liposomes belong to the larger family of pH-sensitiveliposomes, which further comprise pH-sensitive anionic or cationicliposomes, prototypes of which have been presented in Lai et al.,Biochemistry (1985) 24:1654-1661 and Budker et al., Nat. Biotech. (1996)14:760-764. Unlike the pH-sensitive anionic or cationic liposomes,amphoteric liposomes are complex structures and comprise at least a pairof lipids having complementary charge. WO02/066012 discloses a keyfeature of amphoteric liposomes in that these have a stable phase atboth low and neutral pH. WO 02/066012 and WO07/107304 describe a methodof loading such particles with nucleic acids starting from a low pH.

Hafez, et al. (Biophys. J. 2000, 79(3), 1438-1446) and WO 02/0666012provide some guidance as to how to select lipid mixtures with trulyamphoteric properties and more specifically how to determine theirisoelectric point and onset of fusion. Neutral lipids can be additionalconstituents of amphoteric liposomes. The inclusion of one or more suchneutral lipids significantly adds to the complexity of the mixture,especially since the individual amounts of all the components may vary.The very high number of possible combinations of lipids represents apractical hurdle towards a more rapid optimisation of amphotericliposomes. In this regard, WO08/043575 reveals strategies for theoptimization of stability, fusogenicity and cellular transfection ofamphoteric liposomes, particularly a method of predicting which mixturesof lipids form satisfactorily stable lamellar phases at high and low pH,whilst forming a fusogenic, hexagonal phase at an intermediate pH.

The amphoteric liposomes according to the abovementioned references arepotent transfectants of cells. However, it was observed that thefunction of some of these liposomes could be blocked by the addition ofcertain sera, thereby potentially limiting the activity of theseliposomes for the targeting of certain cells in vivo. This is furtherillustrated in the Examples presented herein, e.g., Example 3.

The inhibition of the uptake of amphoteric liposomes observed indifferent sera is apparently opposite to the recently publishedactivation of cationic carrier through complex formation withlipoproteins, in this case ApoE, as demonstrated in Akinc et al., Mol.Ther. (2010) electronic publication on May 11th, ahead of print. DOI:10: 1038/mt.2010.85

A more detailed investigation revealed lipoproteins as mediators of thisinhibitory effect. As shown in Example 4 herein, human serum deficientof lipoproteins is no longer able to inhibit the uptake of liposomes asindicated by the functional delivery of siRNA to the challenged cells.The inventors have now surprisingly and unexpectedly found that certainspecies of cationic imino lipids in combination with anionic lipidshaving a carboxyl or phosphate moiety in their polar head groups areparticularly advantageous in maintaining transfection activity in thepresence of serum. Frequently, a particular advantage was observed whenthe lipid assemblies or liposomes created from said lipid mixtures wereformulated according to the method described herein and in WO08/043575.

OBJECT OF THE INVENTION

It was therefore an object of the invention to provide lipid assembliesor liposomes that can transfect cells in the presence of various sera.

Another object of the invention is to provide pharmaceuticalcompositions comprising such liposomes as a carrier for the delivery ofactive agents or ingredients, including drugs such as nucleic aciddrugs, e.g., oligonucleotides and plasmids into cells or tissues.

SUMMARY OF THE INVENTION

The present invention provides lipid assemblies, liposomes and their usefor transfection of cells wherein said lipid assemblies comprise anionicand cationic amphiphiles and wherein at least a portion of the cationicamphiphiles are imino lipids that are substantially charged at pH7.5,and wherein the anionic amphiphiles are carboxyl or phosphate lipids andwherein further the charge ratio between the cationic and anionicamphiphiles is 1.5 or less.

In various embodiments of the invention, lipid assemblies comprisinganionic and cationic amphiphiles are provided wherein at least a portionof the cationic amphiphiles are imino lipids that are substantiallycharged under physiological conditions, and wherein further at least aportion of the anionic amphiphiles are carboxyl lipids, and wherein theratio between the cationic and anionic amphiphiles is lower or equal to1.5.

In more specific aspects of the invention, lipid assemblies comprising acombination of lipids are provided wherein the cationic lipids of saidcombination comprise a guanido moiety and the anionic lipids of saidcombination comprise a carboxyl group, further characterized in that theratio between the guanido moieties and the carboxyl groups is lower orequal to 1.5.

In other embodiments of the invention, lipid assemblies comprisinganionic and cationic amphiphiles are provided wherein at least a portionof the cationic amphiphiles are imino lipids that are substantiallycharged under physiological conditions, and wherein further at least aportion of the anionic amphiphiles are phosphate lipids, and wherein theratio between the cationic and anionic amphiphiles is lower or equal to1.5. In further preferred aspects of such embodiments, the imino lipidsare guanido lipids.

The charged imino groups of the cationic amphiphiles of the inventionshave a pK of greater than 7.5 and are selected from imines, amidines,pyridines, 2-aminopyridines, heterocyclic nitrogen bases, guanidomoieties, isoureas or thioisoureas. In preferred embodiments, thecationic lipids are selected from the group of PONA, CHOLGUA, GUADACA,MPDACA or SAINT-18.

In preferred embodiments, the anionic lipids are selected from the groupof CHEMS, DMGS, DOGS, DOPA or POPA.

In many embodiments, the lipid assemblies of the invention areliposomes.

In further embodiments, the lipid assemblies also comprise neutrallipids such as cholesterol, phosphatidylcholine,phosphatidylethanolamine or sphingomyelin or mixtures thereof.

In preferred embodiments the neutral lipid is cholesterol and the molarfraction of cholesterol in the lipid mixture is between 10 and 50 mol %.

In some embodiments, the lipid assemblies also comprise PEGylated lipidsand in preferred aspects of such embodiments the liposomes are producedby a process comprising the steps of (i) formation and sealing of theliposomes in the presence of an active ingredient and (ii) a separateaddition of PEG-lipids after said step (i).

It was unexpectedly found that serum resistant transfection can beachieved with lipid assemblies or liposomes having an outer surfacecomprising a mixture of anionic and cationic moieties; wherein at leasta portion of the cationic moieties are imino moieties that areessentially charged under physiological conditions. In numerousembodiments, the lipid assemblies and liposomes of the present inventionare formulated using a method described in WO08/043575 and alsodescribed in more detail herein.

DETAILED DESCRIPTION OF THE INVENTION

Lipid Chemistry

By “chargeable” is meant that the amphiphile has a pK in the rangebetween 4 to pH 8. A chargeable amphiphile may therefore be a weak acidor base. “Stable” in connection with charged amphiphiles means a strongacid or base with a pK outside this range, which results insubstantially stable charge on the range pH 4 to pH 8.

By “amphoteric” herein is meant a substance, a mixture of substances ora supra-molecular complex (e.g., a liposome) comprising charged groupsof both anionic and cationic character wherein:

1) at least one, and optionally both, of the cation and anionicamphiphiles is chargeable, having at least one charged group with a pKbetween 4 and 8.

2) the cationic charge prevails at pH 4, and

3) the anionic charge prevails at pH 8.

As a result the substance or mixture of substances has an isoelectricpoint of neutral net charge between pH 4 and pH 8. Amphoteric characteris by this definition different from zwitterionic character, aszwitterions do not have a pK in the range mentioned above. Inconsequence, zwitterions are essentially neutrally charged over a rangeof pH values; phosphatidylcholines and phosphatidylethanolamines areneutral lipids with zwitterionic character.

By “charge ratio” or “C/A” herein is meant the absolute value or modulusof the ratio between the nominal charges usually assigned to thecationic and anionic amphiphiles, respectively. The nominal charge of acarboxyl group is “−1”, that of a phosphate moiety is “−2” and thenominal charge of an imino compound is “+1”. The “charge ratio” in agiven mixture of amphiphiles or in a lipid assembly is then calculatedfrom the product of these nominal charges and the respective molarfractions of the compounds considered, neutral compounds such ascholesterol or zwitterionic amphiphiles such as POPC or DOPE are nottaken into account.

C/A=(x _(c1) *z _(c1) +x _(c2) *z _(c2) + . . . x _(cn) *z _(cn))/(x_(a1) *z _(a1) +x _(a2) *z _(a2) + . . . x _(an) *z _(an))

Wherein x_(c1 . . . n) represents the molar fraction of a given cationiccompound, x_(a1 . . . n) represents the molar fractions of anioniccompounds, z_(c1 . . . n) stands for the nominal charge of a givencationic compound and z_(s1 . . . n) represents the nominal charge ofthe anionic compound.

As an example, a mixture comprising 42 mol % of a carboxyl lipid, 38% ofan imino lipid and 20 mol % of a neutral lipid has a charge ratio or C/Aof 38/42=0.91. Another mixture comprising 27% of a phosphate lipid, 43mol % of an imino lipid and 30 mol % of a neutral lipid has a chargeratio or C/A of 43/54=0.8 due to the double nominal charge of thephosphate group.

It becomes apparent from the definition and examples, that molar ratiosor—for the sake of brevity—ratios between lipids and charge ratios havethe same meaning for single-charged species and that these terms can bemutually exchanged within that group. This is for example the case forcombinations of imino and carboxy lipids. In contrast to that, the molarratio is different from the charge ratio for phoshpate lipids, sincethese compounds may bear a double charge, e.g. in cases where thephosphate group is present as a primary phosphate ester as in DOPA. Asshown in the calculation example above, the molar ratio or lipid ratiois then double the charge ratio. For the sake of clarity only, the term“charge ratio” is used with preference throughout this disclosure.

By “physiological pH” or “physiological conditions” herein is meant a pHof about 7.5.

Anionic lipids comprising carboxyl moieties in their polar head groupsare well known to the skilled artisan. Examples of anionic lipidscomprising carboxyl moieties in the polar head groups can be selectedfrom the structures (1)-(4) below,

wherein n or m is an integer between 0 and 29, R₁ and R₂ areindependently from each other an alkyl, alkenyl or alkinyl moietieshaving between 8 and 24 carbon atoms and 0, 1 or 2 unsaturated bonds, A,B or D are independently from each other absent, —CH2—, —CH═, ═CH—, —O—,—NH—, —C(O)—O—, —O—C(O)—, —C(O)—NH—, —NH—C(O)—, —O—C(O)—NH—,—NH—C(O)—O—, a phosphoric or phosphorous acid diester, and “sterol” canbe a cholesterol attached via its C3 atom.

The list below provides further specific examples of lipids carrying acarboxyl group.

CHEMS Cholesterolhemisuccinate Chol-COOH Cholesteryl-3-carboxylic acidor Chol-C1 Chol-C2 Cholesterolhemioxalate Chol-C3Cholesterolhemimalonate Chol-C3N N-(Cholesteryl-oxycarbonyl)glycineChol-C5 Cholesterolhemiglutarate Chol-C6 Cholesterolhemiadipate Chol-C7Cholesterolhemipimelate Chol-C8 Cholesterolhemisuberate Chol-C12Cholesterolhemidodecane dicarboxylic acid Chol-C13N12-Cholesteryloxycarbonylaminododecanoic acid Chol-C16Cholesterolhemihexadecane dicarboxylic acid

Cholesterolhermidicarboxylic acids andCholesteryloxycarbonylaminocarboxylic acids of following generalformula:

wherein Z is C or —NH— and n is any number between 0 and 29.

DGS or Diacylglycerolhemisuccinate (unspecified DG-Succ membrane anchor)DOGS or Dioleoylglycerolhemisuccinate DOG-Succ DMGS orDimyristoylglycerolhemisuccinate DMG-Succ DPGS orDipalmitoylglycerolhemisuccinate DPG-Succ DSGS orDistearoylglycerolhemisuccinate DSG-Succ POGS or1-Palmitoyl-2-oleoylglycerol-hemisuccinate POG-Succ DOGMDioleoylglycerolhemimalonate DOGG Dioleoylglycerolhemiglutarate DOGADioleoylglycerolhemiadipate DMGM Dimyristoylglycerolhemimalonate DMGGDimyristoylglycerolhemiglutarate DMGA DimyristoylglycerolhemiadipateDOAS 4-{(2,3-Dioleoyl-propyl)amino}-4-oxobutanoic acid DOAM3-{(2,3-Dioleoyl-propyl)amino}-3-oxopropanoic acid DOAG5-{(2,3-Dioleoyl-propyl)amino}-5-oxopentanoic acid DOAA6-{(2,3-Dioleoyl-propyl)amino}-6-oxohexanoic acid DMAS4-{(2,3-Dimyristoyl-propyl)amino}-4-oxobutanoic acid DMAM3-{(2,3-Dimyristoyl-propyl)amino}-3-oxopropanoic acid DMAG5-{(2,3-Dimyristoyl-propyl)amino}-5-oxopentanoic acid DMAA6-{(2,3-Dimyristoyl-propyl)amino}-6-oxohexanoic acid DOP2,3-Dioleoyl-propanoic acid DOB 3,4-Dioleoyl-butanoic acid DOS5,6-Dioleoyl-hexanoic acid DOM 4,5-Dioleoyl-pentanoic acid DOG6,7-Dioleoyl-heptanoic acid DOA 7,8-Dioleoyl-octanoic acid DMP2,3-Dimyristoyl-propanoic acid DMB 3,4-Dimyristoyl-butanoic acid DMS5,6-Dimyristoyl-hexanoic acid DMM 4,5-Dimyristoyl-pentanoic acid DMG6,7-Dimyristoyl-heptanoic acid DMA 7,8-Dimyristoyl-octanoic acidDOG-GluA Dioleoylglyceral-glucoronic acid (1- or 4-linked) DMG-GluADimyristoylglycerol-glucoronic acid (1- or 4-linked) DO-cHADioleoylglycerolhemicyclohexane-1,4-dicarboxylic acid DM-cHADimyristoylglycerolhemicyclohexane-1,4-dicarboxylic acid PSPhosphatidylserine (unspecified membrane anchor) DOPSDioleoylphosphatidylserine DPPS Dipalmitoylphosphatidylserine MAMyristic Acid PA Palmitic Acid OA Oleic Add LA Linoleic Acid SA StearicAcid NA Nervonic Acid BA Behenic Acid POGA Palmitoyl-oleoyl-glutamicacid DPAA Dipalmitoylaspartic acid

Any dialkyl derivatives of the anionic lipids comprising diacyl groupslisted above are also within the scope of the present invention.

Preferred anionic lipids having a carboxyl group can be selected fromthe group of Chol-C1 to Chol-C16 including all its homologues, inparticular CHEMS. Also preferred are the anionic lipids DMGS, DPGS,DSGS, DOGS, POGS.

Anionic lipids comprising phosphate moieties in their polar head groupsare well known to the skilled artisan. Examples for phosphate lipids canbe selected from structures (P1)-(P4) below:

wherein n or m is an integer between 0 and 29, R₁ and R₂ areindependently from each other an alkyl, alkenyl or alkinyl moietieshaving between 8 and 24 carbon atoms and 0, 1 or 2 unsaturated bonds, A,B or D are independently from each other absent, —CH2—, —CH═, ═CH—, —O—,—NH—, —C(O)—O—, —O—C(O)—, —C(O)—NH—, —NH—C(O)—, —O—C(O)—NH— or—NH—C(O)—O— and “sterol” can be a cholesterol attached via its C3 atom.

The list below provides further specific examples of lipids carrying aphosphatidic acid group.

Chol-P Cholesterol-3-phosphate DOPA Dioleoyl-phosphatidic acid POPAPalmitoyl-oleoyl-phosphatidic acid DPPA Dipalmitoyl-phosphatidic acidDMPA Dimyristoylphosphatidic acid.

Cetylphosphate or phosphoric acid ester homologues with R1 havingbetween 16 and 24 carbon atoms.

The cationic lipids that can be used with this invention are amphipathicmolecules comprising an imino moiety in their polar head group, whereinsuch imino moiety is substantially charged under physiologicalconditions. Therefore, in preferred embodiments the pK value of thisfunctional group is 7.5 or greater, in further preferred forms the pKvalue of the imino group is 8.5 of higher. Imino moieties having suchcharacteristics can be imines itself or be part of larger functionalgroups, such as amidines, pyridines, 2-aminopyridines, heterocyclicnitrogen bases, guanido functions, isoureas, isothioureas and the like.

The following structures (I1) . . . (I113) represent some specificexamples of such imino moieties,

wherein L represents the apolar region and optionally linker or spacermoieties of the amphipathic lipid molecular. Examples of L can furtherbe selected from the following general structures (11) to (15),

wherein n or m represent an integer between 0 and 29, R₁ and R₂ areindependently from each other an alkyl, alkenyl or alkinyl moietieshaving between 8 and 24 carbon atoms and 0, 1 or 2 unsaturated bonds, A,B or D are independently from each other absent, —CH2—, —CH═, ═CH—, —O—,—NH—, —C(O)—O—, —O—C(O)—, —C(O)—NH—, —NH—C(O)—, —O—C(O)—NH— or—NH—C(O)—O— and “sterol” can be a cholesterol attached via its C3 atom.

The following Table 1 provides calculated or database values for the pKof the imino containing moieties (I1) through to (I113). Forquarternized imino moieties, a hypothetical value of 99 was introducedto merely highlight this fact.

TABLE 1 pK values for the moieties I1-I113 pK moiety imino amino ring Nguanido N I1 10.49 I2 7.23 I3 7.23 I4 7.08 I5 8.41 I6 8.06 I7 7.87 I87.52 I9 11.58 I10 6.18 I11 6.61 I12 7.01 I13 n.d. I14 n.d. I15 5.62 I165.89 I17 0.63 I18 4.53 I19 6.22 I20 6.99 I21 5.36 I22 5.11 I23 5.85 I246.03 I25 12.06 −5 I26 12.37 −4.91 I27 12.37 −4.91 I28 12.37 −4.91 I2912.37 −3.58 I30 12.37 −3.68 I31 12.37 −3.58 I32 12.06 −5 I33 12.68 −3.49I34 12.66 −3.58 I35 10.98 −5.43 I36 12.98 −4.25 I37 12.52 −3.12 I3812.82 −4.01 I39 13.13 −3.93 I40 13.12 −3.68 I41 12.37 −3.25 I42 12.68−4.04 I43 12.99 −3.96 I44 12.98 −3.71 I45 9.1 −4.89 I46 9.37 −3.47 I4710.66 −3.56 I48 99 −3.47 I49 8.47 −4.89 I50 9.02 −3.47 I51 10.31 −3.56I52 99 −3.47 I53 7.73 I54 10.62 −6.91 I55 1.92 −5.58 I56 10.63 −6.87 I578.62 −7.89 I58 11.03 −5.39 I59 9.31 −4.75 I60 8.67 −6.83 I61 9.37 −3.47I62 10.66 −3.56 I63 99 −3.47 I64 7.19 −7.59 I65 7.41 −2.85 I66 8.37−2.58 I67 99 −2.7 I68 13.72 −1.04 I69 14.03 2.05 I70 14.14 1.71 I7111.11 0.94 I72 14.33 1.68 I73 14.25 −0.71 I74 14.73 −0.4 I75 13.9 −0.09I76 14.04 −0.1 I77 14.18 −0.72 I78 14.67 −0.41 I79 14.18 −0.2 I80 14.33−0.2 I81 9.85 −1.92 I82 10.17 −0.57 I83 11.41 −0.65 I84 99 −0.57 −13.15I85 14.33 −0.98 I86 14.33 −0.57 I87 14.47 −0.68 I88 99 −0.57 −11.28 I8910 −8.4 I90 8.69 −9.2 I91 10.93 −7.8 I92 10.08 −6.76 I93 10.32 −6.88 I943.51 I95 3.51 I100 8.98 −8.16 I101 8.85 −8.94 I102 9.9 −7.55 I103 9.69−6.76 I104 9.29 −6.88 I105 8.82 −9.73 I106 10.58 −8.09 I107 12.49 −3.67I108 12.49 −3.67 I109 12.36 −3.67 I110 12.8 −3.58 I111 12.78 −3.58 I11210.62 −3.58 I113 10.27 −3.58

It becomes apparent from the data presented here, that most of thestructures I1-I113 comprise preferred imino moieties having a pK greater7.5 or even greater than 8.5.

The pK values can be taken from public databases. Alternatively, thereis expert software in the public domain that can calculate, predict orextrapolate such values, e.g., ACD/Labs v7 (by Advanced ChemistryDevelopment, Ontario, Canada) or the like.

The imino moieties analyzed above are illustrating the teachings of thisinvention, without limiting it to the specific examples. It is of coursepossible to change the position of substituents, in particular when ringsystems such as pyrrols or pyridins are used for practicing thisinvention. It is also possible to replace the aliphatic radicals usedthroughout I1-I113 with aromatic residues or aryl moieties. Thefollowing list of compounds (A1) through to (A21) provides a fewexamples that should further illustrate such modifications, wherein L isdefined as above.

The following Table 2 provides calculated or database values for the pKof the imino containing moieties (A1) through to (A21)). Forquarternized imino moieties, a hypothetical value of 99 was introducedto merely highlight this fact.

TABLE 2 pK values for structures (A1) to (A21). structure atom pK atompK atom pK A1 ring 7.29 out −7.16 A2 ring 99 A3 ring 99 out −6.76 A4ring 7.06 out −6.91 A5 ring 4.74 A6 imino 12.15 amidin −4.95 A7 imino3.07 amidin −12.14 ring 99 A8 imino 14.24 ring −1.31 A9 imino 14.18amidin −0.72 A10 imino 12.52 amidin −3.12 A11 imino 14.18 ring −1.27 A12imino 14.25 amidin −0.71 A13 imino 12.31 amidin −5 A14 imino 13.75amidin −0.76 A15 imino 10.98 amidin −5.43 A16 imino 7.96 A17 imino 9.44amidin −8.39 A18 imino 9.78 amidin 0.95 A19 imino 8.52 out −1.86 A20imino 11.97 amidin −6.3 A21 imino 12.5 amidin −3.6

Again, many of the structures presented in the above Table 2 comprisepreferred imino moieties having a pK greater 7.5 or even greater than8.5.

As mentioned above, the charged imino moieties can be combined withlipid anchors or hydrophobic portions to yield lipids or amphiphilesthat are capable of forming lipid bilayers by themselves or can beintegrated into lipid membranes formed from other lipids or amphiphiles.In some embodiments, specific lipids or amphiphiles are selected fromthe examples L1 to L17 presented below.

wherein R₁ and R₂ are independently from each other an alkyl, alkenyl oralkinyl moieties having between 8 and 24 carbon atoms and 0, 1 or 2unsaturated bonds.

Some of these lipids have been presented earlier in the literature, forexample the guanido lipids in WO91/16024, WO97/43363, WO98/05678,WO01/55098, WO2008/137758 (amino acid lipids), in EP 0685234 (based ondiacylglycerols), U.S. Pat. No. 5,965,434 (also based ondiacylglycerols) or the pyridinium compounds in U.S. Pat. No. 6,726,894.Furthermore, as demonstrated in WO29086558 or illustrated in structure(15), it is also possible to use alternative lipid backbones, e.g. thosecomprising a dioxolane linker segment while maintaining thefunctionality of the respective head groups.

Lipid Mixtures and Optional Other Lipids

The present invention discloses lipid mixtures comprising anionic andcationic amphiphiles; wherein at least a portion of the cationicamphiphiles are imino lipids that are substantially charged underphysiological conditions, and wherein further at least a portion of theanionic amphiphiles are carboxyl lipids or phosphate lipids.

A co-presence of both cationic lipids comprising a charged imino moietyin their polar head group and anionic lipids comprising a carboxyl orphosphate function in their polar head group is a central feature ofthis invention. That is, liposomes or lipid assemblies thatsubstantially lack one of these elements are not contemplated in thepractice of the present invention. The cationic imino lipids and theanionic lipids can be present in different ratios; said ratios arecharacterized herein as “charge ratios” (cation:anion ratios, C/A, seedefinitions) throughout this disclosure. In many embodiments the C/Aratio is above 0.33, in preferred embodiments this ratio is above 0.5and in some embodiments the ratio is equal or above 0.66. In preferredaspects of said embodiments the C/A is equal or below 3, in furtherpreferred aspects the ratio is equal or below 2 and in particularlypreferred aspects the ratio is equal or below 1.5.

In many aspects of said embodiments, the resulting lipid mixture hasamphoteric character. Imino lipids having a pK of more than 7.5, andeven more so the preferred imino lipids having a pK of 8.5 or higher areessentially charged under physiological conditions, their actual chargebecomes close and eventually identical with their nominal charge. Thetypical pK of carboxyl lipids is between 4.5 and 6 and these lipids aretherefore also charged at physiological pH. Mixtures of both the iminoand the carboxyl lipid therefore have net negative charge atphysiological pH whenever C/A is smaller than 1, the net charge become 0at C/A=1 and positive for C/A>1.

At low pH, the anionic charge disappears around the pK of the carboxyllipid, which renders lipid mixtures having a C/A<1 first neutral andthen positively charged. The charge reversal is characteristic for C/A<1and defines the amphoteric character. Lipid mixtures having C/A=1 orC/A>1 also undergo a reduction of negative charges at low pH, but nocharge reversal. It should however be noted, that the relationshipbetween C/A and amphoteric character of the resulting lipid assembliesimplies a statistic, essentially equal distribution of the chargedmoieties across a given bilayer. That means that the inner and outerleaflet of a membrane must have the same composition of charged lipidsto maintain the full validity of these calculations. This may not alwaysbe the case as demonstrated in example 9 and liposomes of amphotericcharacter can be formed even with lipid mixtures having C/A>1. Still,the correlations between membrane composition and amphoteric characterdisclosed here give good guidance for the selection of lipid mixtures.

The lipid mixtures may further comprise additional cationic, anionic,neutral/zwitterionic, or functionalized lipids. Additional cationiclipids may be known components such as DOTAP, DODAP, DC-Chol and thelike. Additional anionic lipids ay be selected from negatively chargedphospholipids, such as phosphatidylglycerol, phosphatidic acid,dicetylphosphoric acid, cardiolipin and the like. Neutral orzwitterionic lipids are cholesterol, phosphatidylethanolamine,phosphatidylcholine, sphingomyelin and the like.

In preferred embodiments the neutral lipid is cholesterol. Furtherpreferred are variants wherein the lipid mixtures comprise between 10mol % and 50 mol % of cholesterol, even more preferred are variants withabout 20 mol % and 40 mol % cholesterol.

An group of functionalized lipids are those comprising polymerextensions such polyethylenglycol (PEG-lipids). Numerous PEGylatedlipids are known in the state of the art and essential differences canbe found in (i) the size and degree of branching of the PEG-chain, (ii)the type of the linker group between PEG and the membrane-insertedportion of the molecule and (iii) the size of the hydrophobic, membraneinserted domain of a PEGylated lipid. Further aspects of PEGylation are(iv) the density of the modification in the lipid assemblies and (v)their orientation within such lipid assemblies.

In many embodiments of the aspect (i), the PEG fragment has a molecularweight between 500 Da and 5,000 Da, in more preferred embodiments, thisfragment has a molecular weight of about 700 Da to 2,500 Da and evenmore preferred are PEG fragments of about 2000 Da. In many suchembodiments, the PEG moiety is a single chain, non-branched PEG.

Typical embodiments of aspect (ii) are phosphoethanolamine moieties,diacylglycerols moieties or the polar head groups of ceramides.

The size of the hydrophobic, membrane inserted domain characterized inaspect (iii) is a further important feature of such molecules as itdetermines the membrane residence time of the PEG lipid within abilayer. As an example, PEGylated lipids having a short hydrophobicdomain such as DMPE-PEG2000 (a dimyristoylphosphatidylethanolamine-PEGconjugate, wherein the PEG chain has a molecular weight of 2000 Da)diffuse from a given membrane within seconds, whereas the DSPE-PEG2000homologue resides in a bilayer for many hours or days (see Silvius, J.R. and Zuckermann, M. J. (1993) Biochemistry 32, 3153-3161 or Webb, M.S. et al (1998) in Biochim Biophys Acta 1372: 272-282 or Wheeler et al.(1999) in Gene Ther 6: 271-281.

PEGylation at the same time provides colloidal stability to liposomes,in particular to combinations of cationic liposomes with anionic nucleicacid cargoes as illustrated in U.S. Pat. No. 5,287,591 but also impairsthe cellular uptake and/or endosomal of liposomes (see Shi, F. et al.(2002) in Biochem. J. 366:333-341). A transient PEGylation is now stateof the art and satisfies the need for both colloidal stability andactivity of the particles.

A further aspect (iv) of PEGylation is the density of such modification,which should be between 0.5 and 10 mol % of the lipid mixture, inpreferred embodiments the degree of PEGylation is about 1 to 4 mol %.

Since PEGylation of a given bilayer stabilizes the lameilar phase of thelipid assembly and impairs lipid fusion associated with the formation ofa hexagonal phase, the amount of residual PEG moieties in a bilayer mustbe minimal. This can be achieved by titration of the required amounts ofPEGlipids. In some embodiments of aspect (v) the liposomes are thusPEGylated on both membrane leaflets and the amount of PEG is minimized.In another variant, PEG removal is as complete as possible. While thisis easily achieved for the PEG lipids associated with the outer bilayer,diffusion is essentially not possible for PEG lipids attached to theinterior of the lipid structure. It is thus a preferred embodiment ofthe aspect (v) of this invention to provide liposomes comprising chargedimino and carboxyl or phosphate lipids further comprising PEGylatedlipids, wherein said PEGylated lipids are essentially situated on theouter surface.

Such liposomes can be characterized by the process of their production,wherein liposomes are formed in a first step and this step alsocomprises encapsulation of cargo molecules. The PEG-lipids are theninserted into the outer bilayer of the prefabricated liposomes in asecond step, e.g. by addition of a micellar solution of PEGylated lipidsto the liposome suspension. In a specific embodiment of such process,the liposomes sequestering nucleic acids are formed by mixing of awatery solution of nucleic acids with an alcoholic solution of lipids.Liposomes entrapping nucleic acids are formed spontaneously and thePEGylated lipids are added in a subsequent step.

With particular advantage, such process can be practiced with amphotericliposomes, as these liposomes already provide colloidal stability andthe time element between liposome formation and PEGylation is lesscritical. The preparation of amphoteric liposomes encapsulating nucleicacids is disclosed in WO 02/066012, its continuation US2007/0252295 orfurther in WO 07/107304.

In a preferred embodiment, amphoteric liposomes comprising imino andcarboxyl or phosphate lipids are PEGylated on their outer surface byproviding the required amounts of PEG lipid together with theneutralization buffer. For that, the PEG lipids can be dissolved in theneutralization buffer. In another embodiment, said liposomes are formedand neutralized and the PEG lipid is added separately after a timeinterval of between 0.1 s and several days. In yet another embodiment,the liposomes are formed and neutralized and the liposome suspension isfurther concentrated and the PEG lipids are added after theconcentration of the materials. In yet another embodiment, the liposomesare formed and neutralized and concentrated and the non-encapsulatednucleic acid is removed and optionally the buffer for the liposomesuspension is exchanged and the PEG lipids are added afterwards. Insummary, the PEG lipids can be added at any time after the formation andclosure of the liposomes.

In other embodiments the liposomes comprising imino and carboxyl orphosphate lipids have pH-sensitive cationic character and are PEGylatedon their outer surface by providing the required amounts of PEG lipidupon formation and closure of said liposomes, following the stepsoutline above. Since pH-sensitive liposomes are more prone to formaggregates in the presence of nucleic acids, a rapid PEGylation ispreferred and the PEG lipids are added immediately upon closure of theliposomes, e.g. between 0.1 s and 1 min after their production.

In contrast to the above methods yielding product liposomes that areessentially PEGylated on their outer surface, presence of PEGylatedlipids during the actual formation of liposomes; that is before thenascent structures close, results in a different product. Althoughstructural data have not yet been obtained, the skilled artisan wouldexpect in such situation that a substantial amount of PEG moieties alsoresides in the inner leaflet of the membrane. This is similar to thesituation of the nucleic acid cargo which also has access to bothleaflet of the nascent liposome and of which a substantial portion canbe detected inside the liposomes, once these have closed.

Lipid Assemblies

The components mentioned herein can be assembled in various structuresknown to the skilled artisan. These can be liposomes comprising one or anumber of individual bilayers, other supramolecular lipid assemblies orvesicles having a sizeable interior volume that provides an aqueousphase. It also can be emulsion droplets or structures in the form oflipoplex assemblies, the latter in many embodiments comprisingelectrostatic complexes between the lipids and nucleic acids. Inpreferred embodiments, these structures are liposomes or vesicles. Inmany embodiments, the liposomes or vesicles have a sizeable aqueousinterior. In many aspects of this invention, an active pharmaceuticalingredient is complexated, encapsulated, sequestered or otherwiseassociated with the lipid assemblies.

Given the large number of useful imino and carboxyl or phosphate andadditional lipids, a very high number of potentially useful combinationsdoes exist, thereby creating a further need for selection andoptimization amongst the many variants. WO08/043575 gives specificguidance and provides a method for the optimization of complex lipidassemblies, specifically for lipid bilayers, as discussed in furtherdetail herein. In brief, the teachings in WO08/043575 demonstrate thatamphoteric lipid mixtures for stable bilayers both under acidic andneutral pH conditions, however, bilayers formed from these lipidmixtures can undergo phase transition and fusion at their isoelectricpoint, which typically is at slightly acidic conditions. WO08/043575discloses the use of moderately sized or small lipid head groups for thecharged lipid components. WO 08/043575 also teaches the use of large orbulky buffer ions to stabilize the lamellar phases at low pH during theloading procedure, as well as the use of large or bulky buffer ions tostabilize the lamellar phases at neutral pH during storage. Inparticular, reference is made to pages 44-57 of WO 08/043575, whichfeature the essential elements cited above. The reference furtherdiscloses the use of neutral lipids bearing a small head group such tomaximize the fusion activity. Typical neutral lipids for improved fusionare cholesterol or DOPE. Specific considerations and optimization rulesfor the neutral lipids are further presented in WO 09/047006, inparticular on pages 63 through to 70.

Altogether, WO 08/043575 or WO 09/047006, together referred to as “theReferences” herein provide rational guidance for the optimization oflipid assemblies. The References are not restricted to amphotericliposomes, but provide a comprehensive model for the structure-activityrelationship of lipid assemblies.

The present invention represents an advance in the art, as it providesoptimized methods of formulating liposomes that are capable ofcircumventing cellular binding, interaction or competition withlipoproteins or other serum components. While the methods taught byReferences provide the information for the necessary fusogenicity oflipid assemblies, they are silent with respect to a prediction of thecellular binding of the liposomes.

Thus, it is an object of the present invention to provide lipidassemblies, lipid mixtures, and liposomes formulated by the methoddisclosed in the Reference in combination with the unexpected propertiesobserved when using an imino lipid that is substantially charged underphysiological conditions is used in combination with an anionic lipidhaving a carboxyl or phosphate, that is, negatively charged moiety.Without wishing to be bound by theory, the novel compositions formulatedherein can better facilitate lipoprotein-like cellular binding anuptake—a feature that is not known in the art.

The lipid mixtures described herein can have amphoteric or pH-sensitivecationic properties, both of which are generally conveyed towards thelipid assemblies or liposomes by the lipids forming them. Chargeproperties can easily predicted as described in WO 02/0666012 for asymmetrical distribution of the lipids towards both leaflets of a lipidmembrane or bilayer. However, in the cases the lipid distribution of theoutermost leaflet may differ from other parts of the assembly.Macroscopically, lipid mixtures comprising charged imino lipids incombination with carboxyl or phosphate lipids having C/A somewhat largerthan 1 may therefore still form liposomes having amphoteric character,as demonstrated in example 9 and FIG. 1

For purposes of in silico optimization and prediction, lipid mixtures ofthe present invention having a C/A<1 are considered amphoteric and canform lipid assemblies categorized as “amphoter l” mixtures according tothe classification of the References. In other embodiments, lipidmixtures are used that C/A=1 or C/A>1; these are pH-sensitive cationiclipid mixtures, that is their charge is neutral or cationic atphysiological pH and become more cationic with descending pH. ThepH-sensitive cationic mixtures of said embodiments do no longer have anisoelectric point as it is the case with their amphoteric counterparts.Still, the structure-activity relationships provided in the Referencesare applicable as these provide a universal understanding of the phasebehavior of lipid assemblies in combination with solute and ionsirrespective of their charge.

For the sake of clarity, lipid mixtures of the present inventioncomprise one or more cationic lipids having an imino group that issubstantially charged at physiological pH, further comprising one ormore anionic lipids having a carboxyl or phosphate group, optionallyfurther comprising neutral lipids.

The amphoteric character of liposomes has further advantages. Thenegative surface charge of such liposomes or lipid assemblies improvesgreatly the colloidal stability of the liposomes in suspension. This isof particular importance in combinations with polyanionic cargoes suchas nucleic acids, which easily produce aggregates with cationicliposomes.

The negative to neutral surface charge of the amphoteric lipidassemblies or liposomes is also advantageous when administering theliposomes in vivo, where it prevents unspecific adsorption on endotheliaor the formation of aggregates with serum components as observed withcationic liposomes (see Santel et al., (2006) in Gene Therapy 13:1222-1234 for endothelial adsorption of cationic liposomes or Andreakoset al., (2009) in Arthritis and Rheumatism, 60:994-1005 for theprevention of aggregate formation with amphoteric liposomes).

Thus, in preferred embodiments, the liposomes of this invention haveamphoteric character. Within this group, it is of advantage to avoidvery low percentages of the cationic component to maintain effectiveloading of the particles with polyanionic cargos, e.g. nucleic acids. Infurther preferred embodiments, the C/A is greater 0.5.

When applied systemic, that is, into the bloodstream, the liposomesundergo a certain distribution within the body. Typical target sites areliver and spleen, but also include the circulating phagocytic cells. Theliposomes also contact the endothelia surrounding the blood vessels andmay transfect these cells. The accumulation of liposomes in inflamedsites and tumors is of particular therapeutic relevance.

The skilled artisan would be aware of methods to direct the distributionof particles towards one or the other site. It is well known thatliposomes having a small diameter of about 150 nm or less can penetratethe liver endothelium, thus gaining access to the hepatocytes and othercells of the liver parenchyme. In aspects where targeting of the liverhepatocytes is of therapeutic interest, the liposomes of this inventionscan be 150 nm or less in diameter, in preferred embodiments, theliposomes can be less than 120 nm in diameter.

It is also well known that particles having a diameter of 100 nm or moreare well recognized by phagocytic cells. Therefore, in embodiments wheremacrophages or dendritic cells constitute the target of interest, theliposomes of this invention are 120 nm or larger. In some embodiments,these liposomes are 150 nm or larger. In other embodiments theseliposomes can be as a large as 250 nm, or up to 400 nm in size.

It has also been described that surface charge may influence thecirculation time, hence the biodistribution of liposomes and it is wellestablished that PEGylation reduces the surface charge and results inprolonged circulation of the liposomes. Prolonged circulation isgenerally thought to maximize the distribution towards tumors.Therefore, in aspects where tumors constitute the target of interest,the liposomes of this invention have a small net surface charge and arecharacterized by a C/A of between 0.67 and 1.5. In preferred embodimentsfor such applications the lipid mixtures forming said liposomes have aC/A between 0.8 and 1.25. Also, the liposomes targeting tumors are ofsmall size. In preferred embodiments such liposomes are smaller than 150nm, in further preferred embodiments the liposomes are smaller than 120nm. In some embodiments, the liposomes further comprise PEG lipids.

Cargoes for the Liposomes of this Invention

The liposomes or lipid assemblies of this invention can sequester orencapsulate at least one active agent. Said active agent may comprise adrug. In some embodiments, said active agent may comprise one or morenucleic acids. In preferred embodiments, the active ingredient consistsof nucleic acids.

Without being limited to such use, the liposomes or lipid assembliesdescribed in the present invention are well suited for use as carriersfor nucleic acid-based drugs, such as for example, oligonucleotides,polynucleotides and DNA plasmids. These drugs are classified intonucleic acids that encode one or more specific sequences for proteins,polypeptides or RNAs and into oligonucleotides that can specificallyregulate protein expression levels or affect the protein structurethrough, inter alia, interference with splicing and artificialtruncation.

In some embodiments of the present invention, therefore, the nucleicacid-based therapeutic may comprise a nucleic acid that is capable ofbeing transcribed in a vertebrate cell into one or more RNAs, which RNAsmay be mRNAs, shRNAs, miRNAs or ribozymes, wherein such mRNAs code forone or more proteins or polypeptides. Such nucleic acid therapeutics maybe circular DNA plasmids, linear DNA constructs, like MIDGE vectors(Minimalistic Immunogenically Defined Gene Expression) as disclosed inWO 98/21322 or DE 19753182, or mRNAs ready for translation (e.g., EP1392341).

In other embodiments of the invention, oligonucleotides may be used thatcan target existing intracellular nucleic acids or proteins. Saidnucleic acids may code for a specific gene, such that saidoligonucleotide is adapted to attenuate or modulate transcription,modify the processing of the transcript or otherwise interfere with theexpression of the protein. The term “target nucleic acid” encompassesDNA encoding a specific gene, as well as all RNAs derived from such DNA,being pre-mRNA or mRNA. A specific hybridisation between the targetnucleic acid and one or more oligonucleotides directed against suchsequences may result in an inhibition or modulation of proteinexpression. To achieve such specific targeting, the oligonucleotideshould suitably comprise a continuous stretch of nucleotides that issubstantially complementary to the sequence of the target nucleic acid.

Oligonucleotides fulfilling the abovementioned criteria may be builtwith a number of different chemistries and topologies. Theoligonucleotides may comprise naturally occurring or modifiednucleosides comprising, but not limited to, DNA, RNA, locked nucleicacids (LNA's), unlocked nucleic acids (UNA's), 2′O-methyl RNA (2′Ome),2′ O-methoxyethyl RNA (2′MOE) in their phosphate or phosphothioate formsor Morpholinos or peptide nucleic acids (PNA's). Oligonucleotides may besingle stranded or double stranded.

Oligonucleotides are polyanionic structures having 8-60 charges. In mostcases, these structures are polymers comprising nucleotides. The presentinvention is not limited to a particular mechanism of action of theoligonucleotides and an understanding of the mechanism is not necessaryto practice the present invention. The mechanisms of action ofoligonucleotides may vary and might comprise inter alia effects onsplicing, transcription, nuclear-cytoplasmic transport and translation.

In a preferred embodiment of the invention, single strandedoligonucleotides may be used, including, but not limited to DNA-basedoligonucleotides, locked nucleic acids, 2′-modified oligonucleotides andothers, commonly known as antisense oligonucleotides. Backbone or baseor sugar modifications may include, but are not limited to,Phosphothioate DNA (PTO), 2′O-methyl RNA (2′Ome), 2′Fluoro RNA (2′F), 2′O-methoxyethyl-RNA (2′MOE), peptide nucleic acids (PNA), N3′-P5′phosphoamidates (NP), 2′fluoroarabino nucleic acids (FANA), lockednucleic acids (LNA), unlocked nucleic acids (UNA), Morpholinephosphoamidate (Morpholino), Cyclohexene nucleic acid (CeNA),tricyclo-DNA (tcDNA) and others. Moreover, mixed chemistries are knownin the art, being constructed from more than a single nucleotide speciesas copolymers, block-copolymers or gapmers or in other arrangements.

In addition to the aforementioned oligonucleotides, protein expressioncan also be inhibited using double stranded RNA molecules containing thecomplementary sequence motifs. Such RNA molecules are known as siRNAmolecules in the art (e.g., WO 99/32619 or WO 02/055693). Other siRNAscomprise single stranded siRNAs or double stranded siRNAs having onenon-continuous strand. Again, various chemistries were adapted to thisclass of oligonucleotides. Also, DNA/RNA hybrid systems are known in theart. Other varieties of siRNA's comprise three-stranded constructswherein two smaller strand hydridize to one common longer strand, theso-called meroduplex or sisiRNA's having nicks or gaps in theirarchitecture.

In another embodiment of the present invention, decoy oligonucleotidescan be used. These double stranded DNA molecules and chemicalmodifications thereof do not target nucleic acids but transcriptionfactors. This means that decoy oligonucleotides bind sequence-specificDNA-binding proteins and interfere with the transcription (e.g.,Cho-Chung, et al., in Curr. Opin. Mol. Ther., 1999).

In a further embodiment of the invention, oligonucleotides that mayinfluence transcription by hybridizing under physiological conditions tothe promoter region of a gene may be used. Again, various chemistriesmay adapt to this class of oligonucleotides.

In a still further alternative of the invention, DNAzymes may be used.DNAzymes are single-stranded oligonucleotides and chemical modificationsthereof with enzymatic activity. Typical DNAzymes, known as the “10-23”model, are capable of cleaving single-stranded RNA at specific sitesunder physiological conditions. The 10-23 model of DNAzymes has acatalytic domain of 15 highly conserved deoxyribonucleotides, flanked by2 substrate-recognition domains complementary to a target sequence onthe RNA. Cleavage of the target mRNAs may result in their destructionand the DNAzymes recycle and cleave multiple substrates.

In yet another embodiment of the invention, ribozymes can be used.Ribozymes are single-stranded oligoribonucleotides and chemicalmodifications thereof with enzymatic activity. They can be operationallydivided into two components, a conserved stem-loop structure forming thecatalytic core and flanking sequences which are reverse complementary tosequences surrounding the target site in a given RNA transcript.Flanking sequences may confer specificity and may generally constitute14-16 nt in total, extending on both sides of the target site selected.

In other embodiments of the invention, aptamers may be used to targetproteins. Apatamers are macromolecules composed of nucleic acids, suchas RNA or DNA, and chemical modifications thereof that bind tightly to aspecific molecular target and are typically 15-60 nt long. The chain ofnucleotides may form intramolecular interactions that fold the moleculeinto a complex three-dimensional shape. The shape of the aptamer allowsit to bind tightly against the surface of its target molecule includingbut not limited to acidic proteins, basic proteins, membrane proteins,transcription factors and enzymes. Binding of aptamer molecules mayinfluence the function of a target molecule.

All of the above-mentioned oligonucleotides may vary in length betweenas little as 5 to 10, preferably 15 and even more preferably 18, and asmay as 50 or 60, preferably 30 and more preferably 25, nucleotides perstrand. More specifically, the oligonucleotides may be antisenseoligonucleotides of 8 to 50 nucleotides length that catalyze RNAseHmediated degradation of their target sequence or block translation orre-direct splicing or act as antagomirs; they may be siRNAs of 15 to 30basepairs length; or they may further represent decoy oligonucleotidesof 15 to 30 basepairs length. Alternatively, they can be complementaryoligonucleotides influencing the transcription of genomic DNA of 15 to30 nucleotides length; they might further represent DNAzymes of 25 to 50nucleotides length or ribozymes of 25 to 50 nucleotides length oraptamers of 15 to 60 nucleotides length. Such subclasses ofoligonucleotides are often functionally defined and can be identical ordifferent or share some, but not all, features of their chemical natureor architecture without substantially affecting the teachings of thisinvention. The fit between the oligonucleotide and the target sequenceis preferably perfect with each base of the oligonucleotide forming abase pair with its complementary base of the target nucleic acid over acontinuous stretch of the abovementioned number of oligonucleotides. Thepair of sequences may contain one or more mismatches within the saidcontinuous stretch of base pairs, although this is less preferred. Ingeneral the type and chemical composition of such nucleic acids is oflittle impact for the performance of the inventive liposomes as vehiclesbe it in vivo or in vitro, and the skilled artisan may find other typesof oligonucleotides or nucleic acids suitable for combination with theamphoteric liposomes of the invention.

In certain aspects and as demonstrated herein, the liposomes accordingto the present invention are useful to transfect cells in vitro, in vivoor ex vivo.

SPECIFIC EMBODIMENTS

Cholesterol Based Lipids

To illustrate the teachings of this invention, cationic derivatives ofcholesterol comprising guanido moieties (charged imino group, CHOL-GUA),imidazol moieties (non-charged imino group, CHIM) or dimethylamino ortrimethyl ammonium moieties (non-imino, but charged groups, DC-CHOL orTC-CHOL) were systematically combined with different anionic lipids.

The anionic lipids used were CHEMS (cholesterol as hydrophobic portion,carboxylic acid charge group), DMGS or DOGS (diacylglycerois hydrophobicportion, carboxylic acid charge group) or DOPA (diacyl glycerol ashydrophobic portion, phosphate ester charge group). For most of thecation/anion combinations, a series of 8 binary mixtures having C/Aratios between 0.33 and 2 was prepared, combinations of the cationiclipids with DOPA were tested at C/A 0.75 and 1. Cholesterol was added toall lipid mixtures to constitute between 20 and 40 mol %, as indicated.

All liposomes were loaded with PLK-1 siRNA, an oligonucleotide capableof inhibiting the production of the cell cycle kinase PLK-1 andsuccessful transfection was measured by inhibition of cell viability ofthe test cells (see also Haupenthal et al., Int. J. Cancer (2007),121:206-210. Unspecific inhibition of the cell viability, that is,cytotoxic effects, were monitored by control preparations comprising anon-targeting siRNA of the same general composition and in the sameamounts.

The transfection of cells was followed in regular cell culture medium orwith the additional presence of 10% mouse serum, a potent inhibitor ofcellular uptake for many amphoteric liposomes. The efficacy oftransfection is expressed as IC50, the concentration needed to achieve a50% inhibition of the cell viability.

The ratio between the IC50 in regular medium and the IC50 upon additionof mouse serum is used as a metric for the inhibition of the cellularuptake by mouse serum. This ratio is 5 or higher for liposomes withoutspecific targeting properties. It is 5 or lower for the liposomes ofthis invention; that is liposomes comprising charged imino groups incombination with negatively charged lipids.

As further demonstrated in examples 14, the best serum-resistanttransfection of HeLa cells can be achieved by combinations of CHOLGUAwith the carboxyl lipid DOGS. Particular good results were obtained inthe presence of less than 40% cholesterol and for mixtures having a C/Aof between 0.5 and 1.5. If all other components such as DOGS orcholesterol were kept constant and the GUA head group was exchangedagainst a dimethylamine as in DC-CHOL, the liposomes are still active inthe absence, but no longer in the presence of mouse serum. The same canbe observed for combinations of CHIM and DMGS.

Combinations of cholesterol-based cationic lipids with the phosphatelipid DOPA resemble the findings in that the best activities wasobserved for the imino lipid CHOLGUA. Also, serum-resistant transfectionof CHOLGUA:DOPA liposomes could be observed, although with substantialinhibition compared to the absence of serum. Combinations for DOPA withCHIM or DC-CHOL did not result in any transfection in the presence ofserum.

DACA-Based Lipids

To further investigate the dependence of the serum resistanttransfection from head group chemistry, the following lipids weresynthesized using a common dialkyl-carboxylic acid (DACA) anchor astheir hydrophobic domain:

Wherein the DACA moiety was obtained by addition of oleyliodide to oleicacid as described in the example 10 and the resulting compound is:

Out of the cationic lipids, GUADACA, MPDACA or BADACA have a chargedimino moiety in their polar head groups. The head group of PDACA isessentially uncharged due to the low pK of the pyridine moiety(calculated pK is 5.9) while the methylated variant results in theformation of the constantly charged pyridinium compound MPDACA. ADACAhas a high enough pK of about 9, but lacks the imino component. However,small amounts of the respective enamine may form from that component asthe amino group is situated in □-position from the amide, allowingmesomeric stabilization of the imine form.

Combinations with the anionic lipids CHEMS, DMGS, DOGS and DOPA wereprepared as described above for the cholesterol based lipids and similarseries of different liposomes having various C/A ratios of between 0.33and 2 (or 0.75 and 1 for the phosphate lipid) were produced.

Also, the liposomes were loaded with siRNA targeting PLK-1 or anunrelated sequence and the transfection properties were tested on HeLacells in the presence or absence of mouse serum.

As further demonstrated in examples 14 and 15, serum-resistanttransfection of HeLa cells can be achieved by combinations of GUADACA orMPDACA with carboxyl lipids or phosphate lipids. In addition, theselipids yield very efficient transfection of PLK-1 siRNA also in theabsence of serum. This implies that there is no activation of theliposomes with serum components as recently described for liposomeshaving a dimethylamino head group (Akinc et al., Mol. Ther. (2010)electronic publication on May 11th, ahead of print. DOI:10.1038/mt.2010.85). Very high levels of carrier activity are alsoobserved for C/A ratios between 0.5 and 1.5 for the combinations withthe carboxylic lipids and for C/A 0.75 or 1 for the phosphate lipids. Inmany of these cases, formulations have amphoteric charge properties.

A lack of methylation of the pyridinium compound MPDACA gives therelated PDACA. While still bearing an imine function, this function isno longer charged as in MPDACA; PDACA is also not active as a cationiclipid for transfection purposes. In yet another variant the aromaticring of the head group was kept, but the charged imine was thenpresented as part of an extra-annular aminide group. This compound wasfound active as a lipid for transfection, e.g. in combinations withCHEMS or DMGS where it also resulted in serum-resistant transfection.

Additional lipids based on Dialkylcarboxylic Acids.

Similar findings have been made using the pyridinium lipid SAINT-18 asdescribed in U.S. Pat. No. 6,726,894 (structure 31).

SAINT-18 was combined with various lipid anions, such as CHEMS, DMGS orDOGS. The ratios of the cationic and anionic lipids were varied in asystematic way and the resulting binary mixtures optionally were furthersupplied with 20 or 40 mol % cholesterol. The individual lipid mixtureswere transformed into liposomes and used for the encapsulation of anactive and control siRNA. When tested on HeLa cells in the presence ofnormal cell culture medium, efficient and specific inhibition of thecell viability was observed for numerous of the tested formulations, asdemonstrated in Example 8. However, none of the liposomes having aC/A>=1 yielded transfection of cells in the presence of mouse serum. Instark contrast, a great many of the amphoteric formulations resisted theserum challenge and did transfect the cells effectively. Furthermore,the effect was specific to the PLK-1 siRNA and much higherconcentrations of liposomes loaded with an unrelated siRNA (SCR) wereneeded to unspecifically inhibit cell proliferation. The best resultswere obtained by using SAINT18 in combination with DMGS. Liposomescomprising SAINT-18 and DMGS, further characterized by C/A<1 aretherefore within the purview of this invention.

Amino Acid Based Lipids

To further illustrate the teachings of this invention, the cationicguanido lipid PONA (palmitoyl-oleoyl-nor-arginine, structure 21) wascombined with various lipid anions such as CHEMS or DMGS. The ratios ofthe cationic and anionic lipids were varied in a systematic way and theresulting binary mixtures optionally were further supplied with 20 mol %cholesterol. The individual lipid mixtures were transformed intoliposomes and used for the encapsulation of an active and control siRNA.When tested on HeLa cells, efficient and specific inhibition of the cellviability was observed for most of the tested formulations, asdemonstrated in Example 5. The activity was not or only marginallyaffected by the presence of human or mouse serum.

In example 6, the anionic lipid CHEMS was combined with derivatives ofPONA, wherein the guanido moiety was substituted by an amino group(PONamine) or an quarternized ammonium group (PONammonium) as shown inthe structures (21) and (23).

Again, the ratios between the anionic and cationic lipid components weresystematically varied and 20% cholesterol was present in all lipidmixtures. The material was formulated into liposomes and used for theencapsulation of active and control siRNA. When tested on HeLa cells,efficient and specific inhibition of the cell viability was observed forall formulations comprising a molar excess of the cationic lipids. Formixtures comprising higher molar amounts of the anionic lipid CHEMS, thebest activity was observed in combinations with PONA, while PONamine;CHEMS combinations were only effective in some cases. ThePONammonium:CHEMS combinations were not effective when an excess of theanionic lipid was used.

Moreover, out of the mixtures comprising an excess of the anionic lipidCHEMS, the transfection activity of the PONA:CHEMS combinations was onlymarginally affected by the presence of human or mouse serum, while theactivity of PONamine:CHEMS combinations was completed suppressed in thepresence of mouse serum. The PONammonium formulations remained inactivein the presence of sera.

Combinations of PONA, PONamine or PONammonium with the phosphate lipidsDOPA were also tested as further described in example 15. Both PONA andPONamine, but not PONammonium resulted in serum-resistant transfectionof HeLa cells.

The combined data support a preferred uptake of lipid combinationscomprising guanido lipids in combination with negatively charged, e.g.carboxyl or phosphate lipids. This may relate to the mechanisticconsiderations made further below. The constant and high activity of theformulations having an excess of the cationic lipid component may be dueto electrostatic interaction between these particles and the cellsurface, which however is unspecific. In line with this view is the factthat the activity of the cationic formulations did not depend on eitherthe nature of the anionic or the cationic lipid.

In further experiments, the guanido lipid PONA was combined with CHEMS,DMGS or DOGS. Again, a systematic variation of the ratios of both theanionic and cationic lipid compound in the respective binary mixtureswas performed and the formulations were further supplied with 0, 20 or40 mol % of cholesterol. When tested as above, the great majority of theformulations were active in inhibiting the cell proliferation of HeLacells with an IC₅₀ being lower than 6 nM (see Example 7). A comparisonbetween the concentrations needed for the efficacy of the active andinactive siRNA, however, revealed substantial differences between theformulations. A measure for such comparison is the ratio between theIC₅₀ values for both siRNA's, here expressed as SCR/PLK ratio. Onlyselected formulations reach values significantly higher than 5. Evenmore preferred formulations have SCR/PLK>=10. All of these preferredformulations can be characterized by their ratio between the cationicand anionic lipid component, which i lower than 1.

The invention identifies specific lipid head group chemistry as criticalfor the uptake into certain cells in the presence of otherwiseinhibitory sera. With preference, amphoteric combinations of anioniclipids comprising carboxyl groups and cationic lipids comprising chargedimino moieties result in the desired properties. In contrast, cationicformulations comprising the same lipids do not depend on a specific headgroup chemistry and are less tolerated by cells.

Lipoprotein Binding

The lipoproteins competing with the transfection of liposomes comprise avariety of structures, according to their density. These are known aschylomicrons, VLDL, LDL, IDL or HDL particles. In the endogenouspathway, chylomicrons are synthesized in the epithelial lining of thesmall intestine and are assembled using ApoB-48, a shorter variant ofthe ApoB gene product. Further exchange of lipoproteins with HDLparticles leads to transfer of ApoC-II and ApoE to the chylomicronparticle, the first mediating the activation of lipoprotein lipase, anenzyme needed for the release of lipids from the particle. Thehydrolyzed chylomicrons form so called remnants which are taken upmainly in the liver via recognition of their ApoE portion. Thesynthesis, maturation, use and recycling of VLDL particles follows thevery same pathway, but starts in the liver and is using the ApoB-100protein as its structure forming unit. Again, ApoE mediates the eventualuptake and recycling of the VLDL-remnants, the so-called lDL particles.(see also http://en.wikipedia.org/wiki/Lipoprotein)

ApoE shares structural homology to the apoliproproteins A and C in thatthey all comprise amphipathic tandem repeats of 11 amino acids.Crystallographic data confirm the existence of extended amphipathichelical structures for ApoA-I and and ApoE fragment and also reveal amixed charge organization on the polar face of these helices. These dataare publicly available from the RCSB Protein Data Bank (available atwww.rcsb.org/pdb/home/home.do) and entry 1AV1.pdb gives the proteinstructure of ApoA-I. The amino acids 129 to 166 of 1lpe.pdb representthe LDL-receptor binding fragment of ApoE. In contrast to their overallsimilarity, the three apolipoproteins display specific deviations whentheir amino acid composition is analyzed. In ApoE, arginine is theprevailing cationic amino acid in the tandem repeats. In contrast, ApoAhas equal amounts of lysine and arginine, while ApoC has an excess oflysine residues.

TABLE 3 Analysis of the amino acid composition in tandem repeats ofrelated apolipoproteins. Sequence data were obtained from Swiss-Protavailable at www.expasy.ch/sprot/sprot-top.html). Sequence ApoAIApoE ApoC-II SwissProt Entry P02647 P02649 P02655 Endpoints 68-26780-255 23-101 lenght 199 175 78 IP 5.55 9.16 4.66 # of lysine 18 8 6 #of arginine 14 25 1 # of histidine 5 1 0 # of aspartic acid 10 8 4 # ofglutamic acid 28 22 7 ApoAI ApoE ApoC-II Lysine (%) 9% 5% 8% Arginine(%) 7% 14%  1% Histidine (%) 3% 1% 0% Aspartic acid (%) 5% 5% 5%Glutamic acid (%) 14%  13%  9%

In summary, the polar surface of natural lipoproteins is covered withapolipoproteins, of which ApoE is a common binding motif for thecellular uptake of these particles. The water-exposed portions of ApoErepresent a mosaic of anionic and cationic charges, wherein the anioniccharges are created from the free carboxyl termini of aspartic andglutamic acid residues. The cationic charges comprise a mixture of aminoand guanido groups with a very few imidazols being present.

In order to emulate the recognition pattern of the ApoE binding cassetteon the surface of liposomes, different alternatives can be followed. Itis possible to synthesize ApoE peptide fragment and graft such peptideson the surface of liposomes. This has been demonstrated by Mims et al.,J Biol. Chem. 269, 20539 (1994); Rensen et al., Mol Pharmacol. 52, 445(1997); Rensen et al., J. Lipid Res. 38, 1070 (1997); Sauer et al.,Biochemistry 44, 2021 (2005) or Versluis et al., J Pharmacol. Exp. Ther289, 1 (1999). However, the high cost associated with peptide synthesisand derivatization call for alternative approaches.

A direct presentation of the required charged moieties using mixtures ofdifferent charged lipids, potentially further comprising neutral lipidswould yield a much simpler structure and eliminate the needs for costlypeptide production an derivatization. A considerable challenge of suchan approach is the planar diffusion of the charged groups within thelipid bilayer; it was heretofore unclear whether the affinity of such aless organized assembly would effectively compete with the affinitiesprovided by the authentic lipoproteins. Moreover, the oppositely chargedlipid headgroups may form salt bridges with each other, while only fewhydrogen bonds between functional groups are detected in the bindingcassette of lipoproteins, e.g. ApoE. This may explain the activity ofimino:phosphate lipid combinations such as GUADACA:DOPA or MPDACA:DOPA.While DOPA provides two negative charges under physiological conditions,steric hindrance disables the formation of a salt from one DOPA and twoGUADACA lipids. As such, in these membranes the negatively charged saltbetween DOPA and GUADACA must co-exist with free GUADACA molecules,thereby facilitating the simultaneous presence of separated anionic andcationic elements in a common lipid assembly.

The theory above is mentioned without limiting the findings of thisinvention. Without wishing to be bound to this particular theory, onecan assume that the combinations of charged imino lipids with negativelycharged carboxyl or phosphate lipids emulate the surface properties oflipoproteins covered with ApoE. The particles can of course be used,developed and optimized without such knowledge. The theoreticalbackground may however be helpful to understand guiding principles orapplicability of the vectors described in the various embodiments ofthis invention.

It is for example known, that lipoprotein receptors have differentexpression profiles in various cell types and such knowledge can be usedto assess target cell populations for the liposomes of this invention.

the LDL-receptor is highly expressed on tumors and on thebronchoepithelial cells of the lung (see Su Al, Wiltshire T, Batalov S,et al (2004). Proc. Natl. Acad. Sci. U.S.A. 101 (16): 6062-7, alsopublished at http://en.wikipedia.org/wiki/File:PBB₁₃GE_LDLR_202068_s_at_tn.png)

The liposomes of this invention are thus specifically suited forapplications in the field of oncology, but also for transfection ofspecific lung cells. While tumors are accessible from systemiccirculation through the EPR-effect (enhanced permeability andretention), that is via leaky tumor vasculature, the bronchoepithelialcells can be targeted also from the airways.

In a specific embodiment of this invention, aerosols from liposomescomprising charged imino and carboxyl or phosphate lipids are thus usedfor inhaled dosage forms for the targeting of lung cells, in particularbronchoepithelial cells.

Figure Legends

FIGS. 1-6 display the results of the screening experiment described inexample 14. The nature of the cationic lipids is indicated in thesmaller figures and other legends and axis are similar for all displayitems and are given in the separate smaller figure below. The doublebars denote liposomes with 20% cholesterol (left bar) and 40%cholesterol (right bar), respectively.

Bars represent the IC50 values for the respective liposome/siRNAcombinations under the experimental conditions for each figure, that is,either in the presence of absence of mouse serum. These IC50 valuesdenote the concentrations needed for a half-maximal inhibition of thecell growth and are given in nM. The maximum concentrations of the testitems were 40 and 36 nM for the absence or presence of mouse serum,respectively.

The order of the test items is as follows:

FIG. 1 the anionic lipid is CHEMS−no addition of mouse serum

FIG. 2 the anionic lipid is CHEMS+addition of mouse serum

FIG. 3 the anionic lipid is DMGS−no addition of mouse serum

FIG. 4 the anionic lipid is DMGS+addition of mouse serum

FIG. 5 the anionic lipid is DOGS−no addition of mouse serum

FIG. 6 the anionic lipid is DOGS+addition of mouse serum

EXAMPLES

The teachings of this invention may be better understood with theconsideration of the following examples. However, these examples shouldby no means limit the teachings of this invention.

Example 1—Liposome Product, Characterization and Encapsulation of siRNA

Liposomes were prepared methods as disclosed in WO07/107304. Morespecifically, lipids were dissolved in isopropanol and liposomes wereproduced by adding siRNA solution in NaAc 20 mM, Sucrose 300 mM, pH 4.0(pH adjusted with HAc) to the alcoholic lipid mix, resulting in a finalalcohol concentration of 30%. The formed liposomal suspensions wereshifted to pH 7.5 with twice the volume of Na₂HPO₄ 136 mM, NaCl 100 mM(pH 9), resulting in a final lipid concentration of 3 mM and a finalisopropanol concentration of 10%.

Liposomes were characterized with respect to their particle size usingdynamic light scattering (MALVERN 3000HSA).

Active siRNA: 21mer blunt ended targeting mouse and human PLK-1 mRNA asin Haupenthal et al., Int. J. Cancer (2007), 121:206-210.

Control siRNA (SCR): 21 mer from the same source.

Example 2—General Cell Culture and Proliferation Assay

HeLa cells were obtained from DSMZ (German Collection of Micro Organismand Cell Cultures and maintained in DMEM (Gibco-Invitrogen) andsupplemented with 10% FCS. The cells were plated at a density of 2.5×10⁴cells/ml and cultivated in 100 μl medium at 37° C. under 5% CO₂. After16 h, the liposomes containing siRNA were diluted and 10 μl were addedto the cells to yield final concentrations between 0.4 to 100 nM Plk1 orscrambled siRNA; 10 μl dilution buffer were also added to untreatedcells and into wells without cells. Cell culture dishes were incubatedfor 72 h at 37° C. under 5% CO₂.

Cell proliferation/viability was determined by using the CellTiter-BlueCell Viability assay (Promega, US) according to the instructions of thesupplier.

Example 3—Inhibition of Transfection by Sera

Liposomes from DODAP:DMGS:Cholesterol (24:36:40 mol %) were loaded withactive and control siRNA as above and 25 μl of the liposomes wereincubated with 75 μl sera from different species (SIGMA-Aldrich) for 30min. Following that, liposomes were added to the cells, incubation wascontinued for 72 h and cell viability was determined as above.

When incubated without serum, administration of the active siRNA resultsin a strong inhibition of cell proliferation. As demonstrated in theTable 7 below, this process is inhibited by the addition of sera.

TABLE 7 Inhibition of cellular transfection by sera of different origin.siRNA Call siRNA type concentration Serum viability (%) PLK1 50 nM no 7PLK1 50 nM Human 98 PLK1 50 nM Hamster 80 PLK1 50 nM Rat 108 PLK1 50 nMMouse 102 No No No 100

Example 4—Inhibition is Lipoprotein Dependent

Liposomes as in Example 3 were incubated with human serum devoid ofcertain complement factors or lipoproteins (SIGMA-Aldrich) as above andanalyzed for their ability to mediate the RNAi effect on HeLa cells.

As demonstrate in Table 8, the efficacy of transfection can be restoredby a depletion of lipoproteins. Removal of complement factors wasineffective.

TABLE 8 Restoration of cellular transfection in sero being deficient ofvarious factors. siRNA Cell siRNA type concentration Serum viability (%)PLK1 50 nM no 7 PLK1 50 nM Human, complete 98 PLK1 50 nM Human, no C3 91complement factor PLK1 50 nM Human, no C9 98 complement factor PLK1 50nM Human, lipoprotein 18 deficient No No No 100

Example 5—Serum Resistant Transfection Using a Quanido Lipid

A series of liposomes was constructed form PONA: ANionicLipid:Cholesterol (x:y:20 mol %) and loaded with active and controlsiRNA as in Example 1. Within that series, the ratio between thecationic component PONA and the anionic lipids CHEMS or DMGS wassystematically varied between 0.33 and 2 as indicated in the table.Liposomes having a ratio of the cationic: anionic lipid of 1 or greaterwere further supplied with 2 mol % DMPE-PEG2000 (Nippon Oils and Fats)to avoid aggregation of the particles. This modification is indicated bya “+” in the table. Control reactions with particles having C/A<1 didnot reveal a change of transfection properties in the presence orabsence of PEG lipids.

HeLa cells were grown and maintained as in Example 2 and sera of humanor mice origin (SIGMA-Aldrich) was added directly to the cells for 120min. Following that, the liposomes were added to the cells inconcentrations between 50 pM and 50 nM, incubation was continued for 72h and cell viability was determined as above. The efficacy oftransfection is expressed here as IC₅₀, the concentration needed toinhibited cell proliferation by 50%. Low IC50 values therefore representhighly effective transfection.

It becomes apparent from the results in the Table 9, that the additionof sera only marginally affects the transfection of siRNA mediated bythe liposomes of the example. Some inhibition is still observed forliposomes from PONA:CHEMS comprising low amounts of the anionic lipid(ratios 0.33 and 0.5, particular strong inhibition with mouse serum).

TABLE 9 Efficacy of transfection of liposomes comprising guanidomoieties in the presence of sera. Ratio cationic anionic lipid 0.33 0.500.67 0.82 1+ 1.22+ 1.5+ 2+ CHEMS No Serum 38.54 1.21 0.40 0.56 1.83 1.610.70 1.42 Human 199.00 2.10 0.62 1.13 2.16 1.92 1.70 1.83 Serum Mouse199.00 50.00 1.56 1.94 2.47 1.90 0.76 1.44 Serum DMGS No Serum 0.23 0.540.01 0.01 Human 1.50 2.39 2.88 2.21 Serum Mouse 0.67 0.69 1.41 1.81Serum

Example 6—Criticality of the Quanido Head Group

Series of liposomes having systematically varied ratios between thecationic and anionic lipid components were produced and loaded withsiRNA as in Example 5. The cationic lipid components were PONA, PONamineand PONammonium, the anionic lipid was CHEMS and the cholesterol contentwas fixed to 20 mol %. Liposomes having a ratio of the cationic: anioniclipid of 1 or greater were further supplied with 2 mol % DMPE-PEG2000(Nippon Oils and Fats) to avoid aggregation of the particles. Thismodification is indicated by a “+” in the table.

Hela cells were grown and maintained as in Example 2 and sera of humanor mice origin (SIGMA-Aldrich) was added directly to the cells for 120min. Following that, the liposomes were added to the cells inconcentrations between 50 pM and 50 nM, incubation was continued for 72h and cell viability was determined as above. The efficacy oftransfection is expressed here as IC₅₀ as in Example 5.

It becomes apparent from the data in Table 10, that only PONA, butneither PONamine and even less so PONammonium mediates the transfectionof HeLa cells in the presence of serum. This is most striking in thecase of mouse serum, which inhibits the transfection more aggressively.An excess of the cationic lipid components to some extent compensate theserum mediated loss of activity, but may be due to unspecificelectrostatic adsorption of these liposomes to the cells.

TABLE 10 Criticality of the guanido head group for the serum resistanttransfection of cells. C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+PONA no serum 42.9 1.8 0.6 1.0 4.1 5.4 2.4 6.8 human serum 80.0 2.5 2.22.0 1.8 2.8 6.2 5.2 mouse serum 80.0 31.1 55.0 5.7 2.1 5.3 8.1 7.5PONamine no serum 3.1 65.0 7.5 100.0 3.0 5.2 3.0 2.5 human serum 100.055.0 11.9 100.0 2.2 2.8 6.1 5.1 mouse serum 70.0 100.0 100.0 100.0 75.070.0 39.3 8.7 PONammonium no serum 80.0 100.0 90.0 90.0 65.0 9.5 9.5 5.2human serum 95.0 90.0 90.0 80.0 90.0 11.8 12.4 15.7 mouse serum 85.0100.0 100.0 100.0 100.0 90.0 75.0 55.0

Example 7—Optimization of the Liposome Composition

Series of liposomes having systematically varied ratios between thecationic and anionic lipid components were produced and loaded withsiRNA as in Example 5. The cationic lipid component was PONA, theanionic lipids were CHEMS, DMGS or DOGS and the cholesterol content wasvaried between 0 and 40 mol %. Liposomes having a ratio of thecationic:anionic lipid of 1 or greater but also some of the otherliposomes were further supplied with 2 mol % DMPE-PEG2000 (Nippon Oilsand Fats) to avoid aggregation of the particles. This modification isindicated by a “+” in the table.

HeLa cells were grown and maintained as in Example 2 and liposomes wereadded to the cells in concentrations between 6 nM and 200 nM, incubationwas continued for 72 h and cell viability was determined as above. Theefficacy of transfection is expressed here as IC₅₀ as in the examplesabove. In addition, the IC₅₀ was determined for the liposomes carryingthe inactive siRNA (SCR) and the ratio between IC₅₀ (PLK1) wasdetermined. A high value for this parameter indicates a very specificinhibition of the cellular viability by the PLK1 siRNA, low unspecificeffects contributed by the carrier and low levels of cytotoxicity ingeneral.

TABLE 11 Optimization results for CHEMS. Lowest and highest detectableIC₅₀ values are 6 and 200 nM, respectively. C/A 0.33 0.33+ 0.5 0.5+ 0.670.67+ 0.82 0.82+ 1+ 1.22+ 1.5+ 2+ PLK  0% Chol 44 77 6 6 6 6 6 6 6 6 6 620% Chol 54 79 6 6 6 6 6 6 6 6 6 6 40% Chol 67 94 6 6 6 6 6 6 6 6 6 6SCR  0% Chol 90 86 113 152 23 200 16 21 15 16 14 11 20% Chol 73 90 109128 200 200 26 23 21 11 16 10 40% Chol 94 117 198 200 200 200 6 6 30 1427 12 SCR/  0% Chol 2.05 1.12 18.86 25.33 3.81 83.33 2.60 3.52 2.50 2.682.30 1.84 PLK 20% Chol 1.37 1.14 18.10 21.39 83.33 83.33 4.26 3.77 3.451.84 2.65 1.69 40% Chol 1.40 1.24 32.96 83.33 83.33 83.33 1.00 1.00 5.002.39 4.48 1.97

TABLE 12 Optimization results for DMGS. Lowest and highest detectableIC₅₀ values are 6 and 200 nM, respectively. C/A 0.33 0.5 0.67 0.82 1+1.22+ 1.5+ 2+ PLK  0% Chol 98 200 200 188 6 6 6 6 20% Chol 6 6 6 6 6 6 66 40% Chol 6 6 6 6 6 6 6 6 SCR  0% Chol 200 200 200 158 14 6 10 14 20%Chol 200 54 8 8 13 9 9 10 40% Chol 155 23 11 6 6 14 9 12 SCR/  0% Chol5.11 no effect no effect 0.84 2.26 1.00 1.66 2.36 PLK 20% Chol 83.339.01 1.27 1.26 2.20 1.55 1.45 1.69 40% Chol 25.85 3.90 1.83 1.00 1.002.27 1.54 1.97

TABLE 13 Optimization results for DOGS. Lowest and highest detectableIC₅₀ values are 6 and 200 nM, respectively. C/A 0.33 0.5 0.67 0.82 1+1.22+ 1.5+ 2+ PLK  0% Chol 200 200 200 200 6 6 6 6 20% Chol 22 200 200200 6 6 6 6 40% Chol 6 170 200 200 6 6 6 6 SCR  0% Chol 200 200 200 20014 10 16 10 20% Chol 200 200 200 200 21 10 12 8 40% Chol 15 197 200 20012 7 9 9 SCR/  0% Chol no effect no effect no effect no effect 2.40 1.592.65 1.63 PLK 20% Chol 22.42 no effect no effect no effect 3.45 1.652.07 1.29 40% Chol 2.48 1.16 no effect no effect 1.93 1.09 1.48 1.55

Example 8—Liposomes Comprising a Pyridinium Lipid

SAINT-18 was used as the cationic lipid, its methylated pyridiniumstructure provides a charged imino moiety. CHEM, DMGS and DOGS wereindividually used as anionic lipids providing the carboxyl functionalgroup. Series of liposomes having systematically varied ratios betweenthe cationic and anionic lipid components were produced and loaded withsiRNA as in Example 5. The lipid mixture was further supplied with 20 or40 mol % cholesterol. Liposomes having a ratio of the cationic:anioniclipid of 1 or greater were further supplied with 2 mol % DMPE-PEG2000(Nippon Oils and Fats) to avoid aggregation of the particles. Thismodification is indicated by a “+” in the table.

HeLa cells were grown and maintained as in Example 2 and liposomes wereadded to the cells in concentrations between 50 pM and 50 nM, incubationwas continued for 72 h and cell viability was determined as above. Theefficacy of transfection is expressed here as IC₅₀ as in the examplesabove. In addition, the IC₅₀ was determined for the liposomes carryingthe inactive siRNA (SCR) and the ratio between IC₅₀ (SCR) and IC₅₀ PLK1)was determined. A high value for this parameter indicates a veryspecific inhibition of the cellular viability by the PLK1 siRNA, lowunspecific effects contributed by the carrier and low levels ofcytotoxicity in general.

TABLE 14 transfection results for liposomes from SAINT-18, CHEMS andcholesterol C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ lipid anionCHEMS, no serum PLK1 20% Chol 2.2 no eff. 1.7 33.9 17.8 7.2 4.1 2.7 40%Chol 7.8 no eff. 1.5 32.0 7.2 4.4 2.1 6.4 SCR 20% Chol no eff. no eff.11.9 no eff. 37.4 19.6 20.7 29.0 40% Chol no eff. no eff. no eff. noeff. no eff. 16.9 18.6 28.0 SCR/PLK-1 20% Chol >22.7 7.2 >1.5 2.1 2.75.0 10.6 40% Chol >6.4 >32.5 >1.6 >7.0 3.9 8.7 4.4 lipid anion CHEMS,plus mouse serum PLK1 20% Chol no eff. no eff. 14.4 no eff. no eff. noeff. no eff. 31.7 40% Chol no eff. no eff. no eff. no eff. no eff. noeff. 35.5 23.1 SCR 20% Chol no eff. no eff. no eff. no eff. no eff. noeff. no eff. no eff. 40% Chol no eff. no eff. no eff. no eff. no eff. noeff. 41.5 38.1 SCR/PLK-1 20% Chol >3.5 >1.6 40% Chol 1.2 1.6

TABLE 15 transfection results for liposomes from SAINT-18, DMGS andcholesterol C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ lipid anionDMGS, no serum PLK1 20% Chol 0.8 2.3 1.7 43.6 24.3 7.5 5.2 3.8 40% Chol1.6 2.3 1.8 2.2 11.4 8.9 3.8 5.8 SCR 20% Chol 7.7 8.2 5.3 36.0 28.1 27.610.5 10.3 40% Chol 4.7 no eff. 22.6 5.7 27.7 28.5 8.1 8.2 SCR/PLK-1 20%Chol 9.2 3.6 3.1 0.8 1.2 3.7 2.0 2.7 40% Chol 2.9 >22.1 12.6 2.5 2.4 3.22.1 1.4 lipid anion DMGS; plus mouse serum PLK1 20% Chol 4.0 8.0 2.7 noeff. 26.5 28.8 no eff. no eff. 40% Chol 2.0 2.2 1.6 1.6 no eff. 21.0 noeff. no eff. SCR 20% Chol 10.1 no eff. 23.4 no eff. 29.1 31.2 25.7 28.440% Chol 7.7 18.0 25.8 6.3 28.0 37.4 31.7 25.7 SCR/PLK-1 20% Chol2.5 >6.2 8.6 1.1 1.1 40% Chol 3.9 8.0 16.5 3.9 1.8

TABLE 16 transfection results for liposomes from SAINT-18, DOGS andcholesterol C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ lipid anionDOGS, no serum PLK1 20% Chol 36.9 38.0 no eff. no eff. 9.2 8.1 7.0 6.140% Chol 6.9 19.4 no eff. no eff. 22.7 8.7 6.6 8.5 SCR 20% Chol no eff.no eff. no eff. no eff. 27.5 20.5 10.2 25.9 40% Chol no eff. no eff. noeff. no eff. no eff. no eff. no eff. no eff. SCR/PLK-1 20%Chol >1.4 >1.3 3.0 2.5 1.5 4.3 40% Chol >7.3 >2.6 >2.2 >5.7 >7.6 >5.9lipid anion DOGS, plus serum PLK-1 20% Chol 2.2 18.4 no eff. no eff.27.5 30.5 26.3 28.1 40% Chol 2.7 7.7 no eff. no eff. 27.4 29.2 30.4 30.8SCR 20% Chol 2.8 no eff. no eff. no eff. 32.6 34.4 30.9 33.2 40% Chol noeff. 8.2 no eff. no eff. 30.6 no eff. no eff. 42.8 SCR/PLK-1 20% Chol1.3 >2.7 1.2 1.1 1.2 1.2 40% Chol >18.6 1.1 1.1 >1.7 >1.6 1.4

As it becomes clear from the data in tables 14 to 16, a large number ofamphoteric liposomes facilitate the transfection of cells even in thepresence of mouse serum. Particularly useful are liposomes comprisingSAINT-18 in combination with the diacylglycerosl DMGS and DOGS, whilethe combination with CHEMS was only effective at C/A=0.67. As with thePONA combinations, the amphoteric constructs transfect the cells withhigh specificity, while the compositions having C/A>1 do not provide ahighly specific transfection as indicated by SCR/PLK1 being below 2.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. The specification and examples shouldbe considered exemplary only with the true scope and spirit of theinvention indicated by the following claims.

Example 9—Zeta Potential Measurements

9.1 Analysis of the Zeta Potential for Liposomes Formed fromPONA:CHEMS:CHOL

100 μl of a lipidmix comprising x Mol % PONA, y mol % CHEMS and 20 Mol %cholesterol (20 mM total lipid concentration, solvent: isopropanol) wasinjected in 900 μl of a buffer comprising 10 mM acetic acid and 10 mMphosphoric acid pH4, X and Y, the molar percentages for PONA and CHEMSwere adjusted to yield the C/A ratios in the 17.

The suspension was immediately vortexed and 3 mL of a pH adjustingbuffer was added. Buffers were selected from the group of: 50 mM aceticacid and 50 mM phosphoric acid, adjusted to pH 4, 5, 6.5 or 7.5 usingNaOH or 50 mM Na₂HPO_(4/)50 mM sodium acetate pH9.4. The mixing pH iswas recorded and is given in the table 17 below together with the zetapotentials of the resulting lipid particles that were monitored using aZetasizer HSA3000.

TABLE 17 Zeta Potentials for lipid particles from PONA:CHEMS:CHOL C/Aratio Final pH 0.5 0.67 0.82 1.00 1.22 1.5 2.00 7.56 −54.40 −58.90−58.20 −61.80 −21.80 22.60 #NV 7.20 −48.47 −46.00 −44.90 −50.00 −21.1014.97 #NV 6.32 −44.33 −37.07 −31.37 0.64 23.43 9.60 #NV 4.84 19.67 18.0022.15 31.80 32.77 32.57 28.37 3.93 35.53 41.73 43.75 46.63 46.20 43.4043.23

Clearly, the particles display amphoteric character even for mixtureshaving a C/A of 1.22, that is, greater than 1. Particles having a C/A of0.67, 0.82 or 1 were also produced at pH7.4 and subsequently exposed tolower pH. There were no apparent changes to the zeta potentials shown intable 17.

9.2 Zeta Potential Measurements for Combinations wherein DOPA is theAnionic Lipid

Lipid particles were also prepared from binary mixture of GUADACA andDOPA, an imino/phosphate combination of lipid head groups. The particleswere prepared in the same fashion as described in 9.1 and the zetapotentials of table 18 were recorded for mixtures having different C/Aratios:

TABLE 18 Zeta Potentials for lipid particles from GUADACA:DOPA C/A finalpH 0.65 0.75 0.98 1.16 1.4 4.5 21 13 38 46 51 5.32 −24 22 20 33 35 6.25−8 −45 −30 2 24 7.02 −61 −67 −8 −56 −6 7.81 −67 −78 −76 −65 −21

As with the particles obtained in 9.1, particles with amphotericcharacter are also obtained with C/A>1. Still, the drift in theisoelectric point follows the expectations.

9.3 Zeta Potential Measurements for DOTAP:CHEMS:CHOL

For comparison, the same measurements were performed with lipid mixtureswherein PONA was substituted by DOTAP. The results are shown in table19. In contrast to PONA:CHEMS, amphoteric particles from DOTAP:CHEMS areonly found at C/A<1.

TABLE 19 Zeta potential for lipid particles from DOTAP:CHEMS:CHOL RatioC/A Final pH 0.67 0.82 1 1.22 7.56 −37.7 −21.83 4.9 13.25 7.20 −50.17−24.1 #NV 12.55 6.32 #NV #NV 11.43 7.37 4.84 25.6 32.1 20.27 9.3 3.9352.13 43.93 47.77 12.15

Example 10—Synthesis of CHOLGUA

25 g cholesterolchloroformiate and 50 equivalents (eq.) ethylendiaminewere dissolved in dichloromethane and allowed to react for 6 h at 20° C.The aminoethylcarbamoyl-cholesterol was isolated using chromatographyand crystallization. Yield was 28.7 g, purity 90%.

CHOLGUA was synthesized from the aminoethylcarboamoyl-cholesterolisolated before, 30 g of the substance were incubated with 1.5eq. of1H-pyrazole-1-carboxamidinium hydrochloride and 4eq.N,N-diisopropylethylamin in dichloromethane/ethanol for 16 h at 20° C.,after which the product was isolated by chromatography. Purity was 95%,Yield 16.5 g.

Example 11—Synthesis of DACA, PDACA and MPDACA

42.4 g of oleyl alcohol, 2.5 eq. of diisoproylazodicarboxylate, 2.5 eq.triphenylphosphine and 5 eq. Lil were reacted in tetrahydrofuran (THF)for 24 h at 20° C. Oleyliodid was isolated by chromatography with apurity of 90%, yield was 13.4 g.

In a second step, 10 g oleic acid were mixed with 2.2 eq. oflithiumdiisopropylamide in THF for 0.5 h at 20° C., after which 1 eq.oleyliodide was added. The mixture was incubated for 2 h at 20° C. andDACA purified from the reaction mix using chromatography. Purity was95%, Yield 14.96 g.

For the synthesis of PDACA, 2 g of DACA, 1.2 eq. of 4-picolylamine, 1.4eq. of O-benzotriazole-1-yl-N,,N′,N′-tetramethyluroniumtetrafluoroborate and 4 eq. of N-methylmorpholine were mixed in THF for24 h at 20° C. The reaction mixture was purified includingchromatography. Purity of PDACA was 95%, yield was 1.72 g.

For the synthesis of MPDACA, 2 g of PDACA was dissolved in THF togetherwith 2eq. of dimethylsulphate and the mixture was incubated for 16 h at20° C., after which MPDACA was purified by chromatography. Purity ofMPDACA: 95%, Yield: 1.71 g.

Example 12—Synthesis of GUADACA

In a first step, 3.5 g DACA and 1.5 eq. of 1,1′-carbonyldiimidazol weredissolved in dichloromethane and incubated for 16 h at 20° C., afterwhich 30 eq. ethylenediamine were added. The reaction mixture wasincubated for 4 h at 20° C. after which aminoethyl-DACA was purifiedincluding chromatography. Purity was 90%, Yield 3.2 g.

GUADACA was synthesized from aminoethyl-DACA and for that, 3.2 g ofaminoethyl-DACA, 2.5 eq. 1H-pyrazole-1-carboxamidine hydrochloride and12eq. N,N-diisopropylethylamine were incubated for 3 h at 20° C., afterwhich GUADACA was isolated. Purity: 95%. Yield: 2.24 g.

Example 13—Synthesis of BADACA

BADACA was synthesized from DACA according to the following procedure:4.15 g DACA, 1.2 eq. p-aminobenzamidine, 1.2 eq.N,N′-dicyclohexylcarbodiimid and 3 eq. of 4-Dimethylaminopyridine weremixed in dry dimethylformamide and incubated for 16 h at 70° C. BADACAwas isolated from the reaction using chromatography. Purity: 95%, Yield:1.62 g

Example 14—Serum Resistant Transfection of DACA or Cholesterol BasedCationic Lipids in Combination with Carboxyl Lipids

Series of liposomes having systematically varied ratios between thecationic and anionic lipid components were produced and loaded withsiRNA as in Example 5. The cationic lipid components were CHOLGUA, CHIM,DC-CHOL, TC-CHOL, GUADACA, MPDACA, BADACA and PDACA. The anionic lipidswere CHEMS, DMGS or DOGS and the cholesterol content was either 20 or 40mol %, all lipid mixtures are identified in the data tables. Liposomeshaving a ratio of the cationic: anionic lipid of 1 or greater (C/A>1)were further supplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils andFats).

HeLa cells were grown and maintained as in Example 2 and mouse serum(SIGMA-Aldrich) was added directly to the cells for 120 min. Followingthat, the liposomes were added to the cells, incubation was continuedfor 72 h and cell viability was determined as above. The highestconcentrations of liposomes were 40 nM and 36 nM for experiments in theabsence or presence of mouse serum, respectively. The efficacy oftransfection is expressed here as IC₅₀ (in nM siRNA) as in Example 5.All results from this screening experiment are shown in FIGS. 1-6.

Many of the transfecting mixtures resulted in very potent transfectionof HeLa cells with siRNA, as indicated by the very low IC50 values.Combinations of lipids comprising imino lipids such as CHOLGUA, but moreso MPDACA, GUADACA or PONA remain potent transfectants even in thepresence of mouse serum.

Example 15—Serum Resistant Transfection of Several Cationic Lipids inCombination with Phosphate Lipid

Series of liposomes having C/A ratios of either 0.75 or 1 were producedand loaded with siRNA as in Example 5. The cationic lipid componentswere CHOLGUA, CHIM, DC-CHOL, GUADACA, MPDACA, BADACA, PONA, DOTAP orDODAP. The anionic lipid was DOPA and the cholesterol content was 40 mol%, all lipid mixtures are identified in table 20. Liposomes were furthersupplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats).

HeLa cells were grown and maintained as in Example 2 and mouse serum(SIGMA-Aldrich) was added directly to the cells for 120 min. Followingthat, the liposomes were added to the cells, incubation was continuedfor 72 h and cell viability was determined as above. The efficacy oftransfection is expressed here as IC₅₀ (in nM of siRNA) as in Example 5.

Many of the transfecting mixtures resulted in very potent transfectionof HeLa cells with siRNA, as indicated by the very low IC50 values.Combinations of lipids comprising imino lipids such as CHOLGUA, but moreso MPDACA, GUADACA or PONA remain potent transfectants even in thepresence of mouse serum.

TABLE 20 IC50 values (nM siRNA) for various liposomes in the presenceand absence of mouse serum. Serum inhibition “not potent” refers to alack of minimum potency in the presence of mouse serum, in these casesthe inhibition factor cannot be defined. The highest concentration ofsiRNA in the test was 146 nM. −mouse +mouse serum serum IC50 IC50 IC50C/A Cation PLK1 IC50 Scr. PLK1 Scr. serum inhibition 0.75 CholGUA 8 160104 146 12 CHIM 26 160 146 146 not potent DC-Chol 28 160 146 146 notpotent MPDACA 5 67 10 146 2 GUADACA 6 39 26 146 4 BADACA 159 160 146 146not potent PONA 6 24 146 146 not potent DOTAP 21 152 146 146 not potentDODAP 160 160 146 146 not potent 1 CholGUA 9 141 128 146 14 CHIM 33 160146 146 not potent DC-Chol 29 160 146 146 not potent MPDACA 12 100 4 1460.3 GUADACA 9 89 7 146 1 BADACA 38 160 146 146 not potent PONA 2 66 21146 10 DOTAP 13 160 76 146 6 DODAP 94 160 146 146 not potent

Example 16—Serum Resistant Transfection is Poor in the Absence ofNegatively Charged Lipids

A series of liposomes was produced from cationic lipids and cholesterolas a neutral lipid. No anionic lipids where used in these preparations.The cationic lipid components were CHOLGUA, CHIM, DC-CHOL, ADACA,GUADACA, MPDACA, BADACA, PONA, DOTAP and DODAP and the liposomes wereproduced with the procedure described in example 5.

The cholesterol content was 40 mol % and liposomes were further suppliedwith 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregateformation in the presence of siRNA.

HeLa cells were grown and maintained as in Example 2 and mouse serum(SIGMA-Aldrich) was added directly to the cells for 120 min. Followingthat, the liposomes were added to the cells, incubation was continuedfor 72 h and cell viability was determined as above. The efficacy oftransfection is expressed here as IC₅₀ (in nM of siRNA) as in Example 5.

The results obtained are shown in table 21 below. In all cases, thetransfection efficacy is substantially lower than that of the mixturesfurther comprising an anionic lipid. With the exception of GUADACA orPONA, there was no activity detectable in the presence of mouse serum.

TABLE 21 IC50 values (nM siRNA) for various liposomes in the presenceand absence of mouse serum. Serum inhibition “not potent” refers to alack of minimum potency in the presence of mouse serum, in these casesthe inhibition factor cannot be defined. The highest concentration ofsiRNA in the test was 160 or 146 nM in the absence of presence of mouseserum, respectively. no mouse serum with mouse serum IC50 IC50 IC50 IC50serum Cation PLK1 Scr. PLK1 Scr. inhibition CholGUA 93 160 146 146 notpotent CHIM 160 160 146 146 not potent DC-Chol 101 109 146 146 notpotent MPDACA 27 154 146 146 not potent GUADACA 22 69 95 146 4 BADACA 99160 146 146 not potent PONA 30 100 70 99 2 DOTAP 160 160 146 146 notpotent DODAP 160 160 146 146 not potent

What is claimed is:
 1. Lipid assemblies comprising anionic and cationicamphiphiles; wherein at least a portion of the cationic amphiphiles areimino lipids that are substantially charged under physiologicalconditions, and wherein the anionic amphiphiles are carboxyl orphosphate lipids and wherein further the charge ratio between thecationic and anionic amphiphiles is 1.5 or less.
 2. Lipid assemblies asin claim 1 comprising anionic and cationic amphiphiles; wherein at leasta portion of the cationic amphiphiles are imino lipids that aresubstantially charged under physiological conditions, and whereinfurther at least a portion of the anionic amphiphiles are carboxyllipids, and wherein the ratio between the cationic and anionicamphiphiles is lower or equal to 1.5.
 3. Lipid assemblies as in claim 1or 2 comprising a combination of lipids wherein the cationic lipids ofsaid combination comprise a guanido moiety and the anionic lipids ofsaid combination comprise a carboxyl group, further characterized inthat the ratio between the guanido moieties and the carboxyl groups islower or equal to 1.5.
 4. Lipid assemblies as in claim 1 comprisinganionic and cationic amphiphilies wherein at least a portion of thecationic amphiphiles are imino lipids that are substantially chargedunder physiological conditions, and wherein further at least a portionof the anionic amphiphiles are phosphate lipids, and wherein the chargeratio between the cationic and anionic amphiphiles is lower or equal to1.5.
 5. Lipid assemblies as in any of the claims 1 to 4, furthercharacterized in that the charged imino groups of the cationicamphiphiles have a pK of greater than 7.5 and are selected from imines,amidines, pyridines, 2-aminopyridines, heterocyclic nitrogen bases,guanido moieties, isoureas or thioisoureas.
 6. Lipid assemblies as inany of the claims 1 to 5, wherein in cationic amphiphiles are selectedfrom the group comprising structures of I1 to I113, structures A1 to A21or structures L1 to L17, wherein members od said group are furtherselected according to a pK greater 7.5.
 7. Lipid assemblies as in any ofthe preceding claims, further characterized in that the cationic lipidsare selected from the group of PONA, CHOLGUA, GUADACA, MPDACA orSAINT-18.
 8. Lipid assemblies as in any of the preceding claims, furthercharacterized in that the anionic lipids are selected from the group ofCHEMS, DMGS, DOGS, DOPA or POPA.
 9. Lipid assemblies as any of thepreceding claims, further characterized in that said assemblies have acharge ratio of the cationic and anionic lipids of between 0.5 and 1.5.10. Lipid assemblies as in any of the claims 1 to 9, furthercharacterized in that said assemblies are liposomes.
 11. Liposomes as inclaim 10, further comprising a neutral or zwitterionic lipid selectedfrom cholesterol, phosphatidylcholine, phosphatidylethanolamine,sphingomyeline or mixtures thereof.
 12. Liposomes as in claim 11,further characterized in that the neutral lipid is cholesterol and themolar fraction of cholesterol in the lipid mixture is between 10 and 50mol %.
 13. Liposomes as in claim 10, 11 or 12, further comprising PEGlipids.
 14. Liposomes as in claim 13, further characterized in that thePEG lipids are situated in the outermost membrane leaflet.
 15. Liposomesas in any of the preceding claims 10 to 14, further comprising an activepharmaceutical ingredient.
 16. Liposomes as in claim 15, wherein saidpharmaceutical ingredient is an oligonucleotide.
 17. Liposomes as inclaim 16, wherein said oligonucleotide is a decoy oligonucleotide, andantisense oligonucleotide, a siRNA, an agent influencing transcription,a ribozyme, DNAzyme or an aptamer.
 18. Liposomes as in claim 17, whereinsaid oligonucleotides comprise modified nucleosides such as DNA, RNA,LNA, PNA, 2′OMe RNA, 2′ MOE RNA, 2′F RNA in their phosphodiester orphosphothioate forms.
 19. The liposomes of claim 14, produced by aprocess comprising the steps of (i) formation and sealing of theliposomes in the presence of an active ingredient and (ii) a separateaddition of PEG-lipids after said step (i).
 20. Use of the liposomes asclaimed in any of claims 10 to 19 for the in vivo, in vitro or ex vivotransfection of cells.
 21. Use of an aerosol comprising liposomes asclaimed in any of the claims 10 to 19 for the transfection of lungcells.